Formulation of HBsAg in Montanide ISA 51VG adjuvant: Immunogenicity study and monitoring long-lived humoral immune responses.

Montanide ISA 51VG adjuvant has been approved for human clinical application and stimulates cellular and humoral immune responses. Here, HBsAg was formulated in Montanide ISA51VG adjuvant to compare its potency with the Fendrix and HBsAg-alum vaccines. In particular, the long-term humoral response was assessed up to 220 days after the final immunization. BALB/c mice were allocated into six groups. Treatment groups were injected with HBsAg-Montanide ISA51VG, the Fendrix and commercial HBsAg-alum, respectively. Montanide ISA51 VG, Alum and PBS injected mice were considered as control groups. Mice were immunized three times with 2-week intervals on days 0, 14 and 28 by subcutaneous injection. Lymphocyte proliferation was assessed with the BrdU method. IFN-γ, IL-2 and IL-4 cytokines, specific total IgG and IgG1/IgG2a isotypes were assessed using ELISA. The HBsAg-Montanide ISA51VG vaccine resulted in a significant increase in lymphocyte proliferation versus HBsAg-alum and higher IL-2 cytokine production versus the Fendrix. Comparable IL-4 and IFN-γ cytokines responses were observed for these vaccines. Following the first immunization, IgG increased more in HBs-Montanide 51VG group versus the HBs-alum group, while after the second and third shots comparable responses were observed in comparison to the HBs-alum group. Monitoring for 220 days after the final vaccination showed the superiority of HBsAg-Montanide ISA 51VG vaccine versus HBsAg-alum and even the Fendrix vaccine in the induction of long-term antibody responses. This study suggests that HBsAg-Montanide ISA51VG as a novel vaccine formulation can trigger both cellular and long-lasting humoral immune responses more efficiently than conventional HBsAg vaccines.

Incomplete Freund's adjuvant reduces arginase and enhances Th1 dominance, TLR signaling and CD40 ligand expression in the vaccine site microenvironment.

BACKGROUND: Immunogenicity of cancer vaccines is impacted by adjuvants and schedule, but systematic assessments of their effects have not been performed. Montanide ISA-51, an incomplete Freund's adjuvant (IFA), is used in many vaccine trials, but concerns have been raised about negative effects in murine studies. We found in humans that IFA enhances systemic immune responses and that repeat vaccination at one site (same site vaccination (SSV)) creates tertiary lymphoid structures (TLS) in the vaccine site microenvironment (VSME). We hypothesized that vaccination with peptides+IFA+pICLC or SSV×3 with peptides in IFA would create an immunogenic milieu locally at the VSME, with activated dendritic cells (DC), TLS-associated chemokines and a Th1-dominant VSME. METHODS: Biopsies of the VSME were obtained from participants on two clinical trials who were immunized with multiple melanoma peptides (MELITAC 12.1) in adjuvants comprising IFA and/or the TLR3-agonist pICLC. Biopsies were obtained either a week after one vaccine or a week after SSV×3. Controls included normal skin and skin injected with IFA without peptides. Gene expression analysis was performed by RNAseq. RESULTS: VSME samples were evaluated from 27 patients. One vaccine with peptides in pICLC+IFA enhanced expression of CD80, CD83, CD86 (p<0.01), CD40 and CD40L (p<0.0001) over normal skin; these effects were significantly enhanced for SSV with peptides+IFA. Vaccines containing pICLC increased expression of TBX21 (T-bet) but did not decrease GATA3 over normal skin, whereas SSV with peptides in IFA dramatically enhanced TBX21 and decreased GATA3, with high expression of IFNγ and STAT1. SSV with peptides in IFA also reduced arginase-1 (ARG1) expression and enhanced expression of TLR adapter molecules TICAM-1 (TRIF) and MYD88. Furthermore, SSV with IFA and peptides also enhanced expression of chemokines associated with TLS formation. CONCLUSIONS: These findings suggest that SSV with peptides in IFA enhances CD40L expression by CD4 T cells, supports a Th1 microenvironment, with accumulation of activated and mature DC. Increased expression of TLR adaptor proteins after SSV with peptides in IFA might implicate effects of the skin microbiome. Reduced ARG1 may reflect diminished suppressive myeloid activity in the VSME. TRIAL REGISTRATION NUMBER: (NCT00705640, NCT01585350).

Protective efficacy of inactivated reverse genetics based equine influenza vaccine candidate adjuvanted with MontanideTM Pet Gel in murine model.

Equine influenza is a leading cause for respiratory illness in equines. Major control measures involve vaccination which requires continuous harmonization owing to antigenic drift. The present study focused on assessing the protective efficacy of an inactivated recombinant equine influenza virus (rgEIV) vaccine candidate adjuvanted with MontanideTM Pet Gel in murine model. The rgEIV was generated using reverse genetics by incorporating HA and NA segments from EIV/H3N8, clade 2-Florida sublineage in an A/WSN/33 /H1N1 backbone and inactivated by formalin. The vaccine was prepared by mixing inactivated rgEIV with MontanideTM Pet Gel adjuvant followed by intranasal inoculation into BALB/c mice intranasally. The immune responses and protective efficacy of the vaccine was evaluated by measurement of antibody titer, immunoglobulin subtyping, cytokines, clinical signs and pathological lesions after immunization and challenge with wild EIV. Serology and cytokine expression pattern indicated that the vaccine activated mixed Th1- and Th2-like responses of vaccine. Booster immunization stimulated strong antibody responses (HAI titre: 192 ± 28.6) at 42 days post immunization and the predominant antibody subtype was IgG1. Upregulation of interferon (IFN)-gamma, interleukin (IL)-12 and IL-2 levels indicates effective induction of Th1 type response. We found that vaccination has protected mice against equine influenza virus challenge as adjudged through a lack of nonappearance of visible clinical signs of disease, no loss of body weight loss, reduced pathology in the lungs and markedly reduced virus shedding from the respiratory tract. Therefore, we conclude that recombinant EIV vaccine candidate adjuvanted with MontanideTM Pet Gel could aid in quick harmonization of the vaccines through replacement of HA and NA genes for control of EIV outbreaks.

Raising antibodies against epoxyscillirosidine, the toxic principle contained in Moraea pallida Bak. (Iridaceae), in rabbits.

Moraea pallida Bak. (yellow tulp) poisoning is the most important plant cardiac glycoside toxicosis in South Africa. The toxic principle, a bufadienolide, is 1α, 2α-epoxyscillirosidine. The aim was to investigate the potential to develop a vaccine against epoxyscillirosidine. Epoxyscillirosidine, proscillaridin and bufalin, were successfully conjugated to hen ovalbumin (OVA), bovine serum albumin (BSA) and keyhole limpet haemocyanin (KLH). There was a low immune response following vaccination of adult male New Zealand White rabbits with epoxyscillirosidine-OVA (n = 3) and OVA (n = 3) using Freund's adjuvant in Trial (T) 1. The immune response improved significantly in T2 following doubling of the dose to 0.8 mg/rabbit and changing the adjuvant to Montanide. In T3, the rabbits (n = 15), allocated into 5 equal groups, vaccinated with proscillaridin-BSA, bufalin-BSA, epoxyscillirosidine-KLH, epoxyscillirosidine-BSA and BSA respectively, using Montanide adjuvant, developed antibodies against the administered immunogens, with epoxyscillirosidine-KLH inducing the highest immune response. Proscillaridin and bufalin antibodies cross-reacted with epoxyscillirosidine in an enzyme linked immunosorbent assay. The conjugation methodology will be adjusted in the future to target optimal conjugation efficiency. Additional vaccination will be conducted in search of neutralizing antibodies against the yellow tulp toxin. The cross-reactivity of proscillaridin and bufalin antibodies with epoxyscillirosidine could be studied in future to explore the potential to prevent yellow tulp poisoning.

Enhanced Immune Responses Against Japanese Encephalitis Virus Infection Using Japanese Encephalitis Live-Attenuated Virus Adjuvanted with Montanide GEL 01 ST in Mice.

Japanese encephalitis virus (JEV) is one of the major causes of acute encephalitis in human and animal. To prevent JEV infection, an effective live-attenuated vaccine is needed. In the article, JEV attenuated strain, SCYA201201 of GI genotype, which was mixed with 10% concentrate GEL 01 ST adjuvant (Montanide™ GEL 01 ST), was selected for a vaccine candidate and its immunogenicity was evaluated in mice. Our results showed that JEV mixed with GEL 01 ST elicited production of both IgG1 and IgG2a antibodies, and enhanced virus-specific crossprotective intergenotypic response in mice. Proliferation of splenocytes was observed in all immunized groups and a relatively higher proliferation activity was detected in JEV mixed with GEL 01 ST group (p < 0.05). In the JEV + 10% GEL 01 ST vaccinated group, the proportion of CD4+ T cells in spleen was significantly higher than that of control group (p < 0.05), and the yields of interleukin (IL)-2, IL-4, and interferon-γ in the splenocyte supernatant were also significantly higher than that of control group (p < 0.05). Moreover, complete protection was provided after JEV challenge in mice in JEV mixed with GEL 01 ST group, and early immunity was detected in those mice immunized with JEV mixed with GEL 01 ST. These findings confirm that GEL 01 ST can enhance JEV live-attenuated immunogenicity, and JEV +10% GEL 01 ST used as vaccine candidates provide protection against JEV infection in a mouse model, which could be used as potential vaccine candidates in pig.

Airway responsiveness to mannitol in asthma is associated with chymase-positive mast cells and eosinophilic airway inflammation.

BACKGROUND: Airway hyperresponsiveness (AHR) to inhaled mannitol is associated with indirect markers of mast cell activation and eosinophilic airway inflammation. It is unknown how AHR to mannitol relates to mast cell phenotype, mast cell function and measures of eosinophilic inflammation in airway tissue. We compared the number and phenotype of mast cells, mRNA expression of mast cell-associated genes and number of eosinophils in airway tissue of subjects with asthma and healthy controls in relation to AHR to mannitol. METHODS: Airway hyperresponsiveness to inhaled mannitol was measured in 23 non-smoking, corticosteroid-free asthmatic individuals and 10 healthy controls. Mast cells and eosinophils were identified in mucosal biopsies from all participants. Mast cells were divided into phenotypes based on the presence of chymase. mRNA expression of mast cell-associated genes was measured by real-time PCR. RESULTS: The proportion of submucosal MCTC was higher in asthmatic individuals with AHR to mannitol compared with asthmatic individuals without AHR (median: 40.3% vs. 18.7%, P = 0.03). Increased submucosal MCTC numbers were associated with increased levels of mRNA for thymic stromal lymphopoietin (TSLP) and CPA3 in asthmatics. Reactivity to mannitol correlated significantly with eosinophils in submucosa (r(s): 0.56, P = 0.01). CONCLUSION: Airway hyperresponsiveness to inhaled mannitol is associated with an altered submucosal mast cell profile in asthmatic individuals. This mast cell profile is associated with increased levels of TSLP and CPA3. The degree of AHR to mannitol is correlated with the degree of eosinophilic inflammation in the airway submucosa.

Evidence for induction of humoral and cytotoxic immune responses against devil facial tumor disease cells in Tasmanian devils (Sarcophilus harrisii) immunized with killed cell preparations.

Tasmanian devils (Sarcophilus harrisii) risk extinction from a contagious cancer, devil facial tumour disease (DFTD) in which the infectious agent is the tumor cell itself. Because devils are unable to produce an immune response against the tumor cells no devil has survived 'infection'. To promote an immune response we immunized healthy devils with killed DFTD tumor cells in the presence of adjuvants. Immune responses, including cytotoxicity and antibody production, were detected in five of the six devils. The incorporation of adjuvants that act via toll like receptors may provide additional signals to break 'immunological ignorance'. One of these devils was protected against a challenge with viable DFTD cells. This was a short-term protection as re-challenge one year later resulted in tumor growth. These results suggest that Tasmanian devils can generate immune responses against DFTD cells. With further optimization of immune stimulation it should be possible to protect Tasmanian devils against DFTD with an injectable vaccine.

Montanide ISA 71 VG is Advantageous to Freund's Adjuvant in Immunization Against S. aureus Infection of Mice.

The enormous capacity of Staphylococcus aureus to acquire antibiotic resistance makes it a permanent task to search for and to develop new anti-infectives. One of the possible approaches is the early active immunization of risk patients and animal stocks to prevent S. aureus infections. Based on a S. aureus proteome screen with S. aureus-specific human antiserum, we have previously identified several anchorless cell wall proteins to be used as novel vaccine candidates. To develop an efficient anti-S. aureus vaccine, the supplemented adjuvants Montanide(™) ISA 71 VG and ISA 206 were compared to Freund's adjuvant in terms of handling, induction of cytokine profile, triggering antigen-specific immunoglobulin production of different IgG subclasses and provision of increased survival rates in our S. aureus sepsis mouse model. Immunization with ISA 71 VG in comparison with Freund's adjuvant induced slightly delayed but comparably strong increase of antigen-specific antibody titres and conferred protective effect against S. aureus challenge. In contrast using ISA 206 as adjuvant, significantly lower IgG titres and consequently, no protective effect against S. aureus infection were observed. Handling and tolerability of the Montanide is superior to Freund's adjuvant. Montanide(™) ISA 71 VG can serve as an effective adjuvant replacement for Freund's adjuvant in research with a prospective usage in animal and human vaccines against bacterial pathogens.

Sex-related differences in pulmonary physiologic outcome measures in a high-risk birth cohort.

BACKGROUND: Sex influences the risk of wheezing illnesses and the prevalence of asthma throughout childhood. OBJECTIVE: To better understand the mechanisms of these effects, we analyzed longitudinal relationships between sex, lung physiology, and asthma in the Childhood Origins of ASThma birth cohort study. METHODS: Childhood Origins of ASThma birth cohort study children were followed prospectively from birth and assessed annually. Results of spirometry, fractional exhaled nitric oxide (Feno), mannitol provocation testing, and (3)He gas magnetic resonance imaging were assessed by sex using multivariate models including age, asthma diagnosis, and wheezing histories. RESULTS: Girls had higher prebronchodilator forced expiratory volume in 0.5 seconds/forced vital capacity values than did boys (mean difference, 0.017; 95% CI, 0.000-0.034; P = .05) of equivalent age. Postbronchodilator findings were more pronounced, with boys demonstrating reduced forced expiratory volume in 0.5 seconds/forced vital capacity values than did girls of equivalent age (mean difference, 0.032; 95% CI, 0.014-0.049; P = .0005). Conversely, girls were noted to have higher ventilation defects on (3)He magnetic resonance imaging than did boys (P = .01). No differences were noted in the rate of positive responses to mannitol provocation or Feno measurements. CONCLUSIONS: Lower airflow values are present by spirometry for prepubertal boys than for age-matched girls; however, greater (3)He ventilation defects were noted in girls. This could represent a greater degree of subclinical air trapping in prepubertal girls because residual volumes are not detected on standard spirometric readings. No differences were noted between the 2 sexes with airway hyperresponsiveness (mannitol provocation testing) or inflammation (Feno). Prospective peripubertal follow-up will determine whether these differences persist or change with the de novo expression and remission of asthma based on sex and age.

Phase I clinical trial of vaccination with LY6K-derived peptide in patients with advanced gastric cancer.

BACKGROUND: Lymphocyte antigen 6 complex locus K (LY6K) has been identified as a tumor-associated antigen in lung cancers and esophageal squamous cell carcinomas. The immunogenicity of LY6K-177 peptide vaccine therapy has been demonstrated in patients with advanced esophageal cancer. This study extends this treatment to gastric cancer. METHODS: LY6K expression in clinical samples obtained from gastric cancer patients was examined by immunochemistry. As a phase I clinical trial, the safety and immunogenicity of LY6K-177 peptide vaccine emulsified with Montanide ISA 51 was evaluated in six patients with unresectable advanced gastric cancer. LY6K-177 peptide (1 mg in 1 ml sterile saline) was emulsified with incomplete Freund's adjuvant (1 ml) and intracutaneously administered to the inguinal region or axilla. One treatment course comprised four vaccinations, performed weekly for the first and second treatment courses and biweekly for the third treatment course. RESULTS: LY6K expression was confirmed in 85 % of gastric cancer tissues. Induration and redness at the vaccination site (grade I), possibly a delayed-type hypersensitivity reaction, was observed in all patients; however, no systemic toxicology was identified in any patient throughout the observation period. Three of the six patients had stable disease, and a tumor contraction effect was observed in one patient. CONCLUSIONS: LY6K was expressed in 85 % of observed gastric cancers. Vaccination with LY6K-177 peptide/Montanide ISA 51 appeared to be tolerated by advanced gastric cancer patients, and moreover anticancer efficacy was suggested. This trial was registered with ClinicalTrial.gov (no. NCT00845611).

Peptide vaccination in Montanide adjuvant induces and GM-CSF increases CXCR3 and cutaneous lymphocyte antigen expression by tumor antigen-specific CD8 T cells.

T cell infiltration of melanoma is associated with enhanced clinical efficacy and is a desirable endpoint of immunotherapeutic vaccination. Infiltration is regulated, in part, by chemokine receptors and selectin ligands on the surface of tumor-specific lymphocytes. Therefore, we investigated the expression of two homing molecules--CXCR3 and CLA - on vaccine-induced CD8 T cells, in the context of a clinical trial of a melanoma-specific peptide vaccine. Both CXCR3 and CLA have been associated with T cell infiltration of melanoma. We demonstrate that a single subcutaneous/intradermal administration of peptide vaccine in Montanide adjuvant induces tumor-specific CD8 T cells that are predominantly positive for CXCR3, with a subpopulation of CXCR3(+)CLA(+) cells. Addition of GM-CSF significantly enhances CXCR3 expression and increases the proportion of CLA-expressing cells. Concurrent with CXCR3 and CLA expression, vaccine-induced CD8 cells express high levels of Tbet, IFN-γ, and IL-12Rß1. Collectively, these studies demonstrate that peptide vaccination in adjuvant induces CD8 T cells with a phenotype that may support infiltration of melanoma.

A multi-peptide, dual-adjuvant telomerase vaccine (GX301) is highly immunogenic in patients with prostate and renal cancer.

BACKGROUND: Anti-tumor vaccination is a new frontier in cancer treatment applicable to immunogenic neoplasms such as prostate and renal cancers. GX301 is a vaccine constituted by four telomerase peptides and two adjuvants, Montanide ISA-51 and Imiquimod. OBJECTIVE: The aim of this study was to analyze safety and tolerability of GX301 in an open-label, phase I/II trial. Immunological and clinical responses were also evaluated as secondary endpoints. EXPERIMENTAL DESIGN: GX301 was administered by intradermally injecting 500 µg of each peptide (dissolved in Montanide ISA-51) in the skin of the abdomen. Imiquimod was applied as a cream at the injection sites. The protocol included 8 administrations at days 1, 3, 5, 7, 14, 21, 35, 63. Eligible patients were affected with stage IV prostate or renal cancer resistant to conventional treatments. Patients were clinically and immunologically monitored up to 6 months from the first immunization. RESULTS: No grade 3-4 adverse events were observed. Evidence of vaccine-specific immunological responses was detected in 100 % of patients. Disease stabilization occurred in 4 patients. Prolonged progression-free survival and overall survival were observed in patients showing a full pattern of vaccine-specific immunological responses. CONCLUSION: GX301 demonstrated to be safe and highly immunogenic. Further studies are needed to determine its clinical efficacy.

Effect of Montanide and poly-ICLC adjuvant on human self/tumor antigen-specific CD4+ T cells in phase I overlapping long peptide vaccine trial.

Vaccination of patients with ovarian cancer with overlapping long peptides (OLP) from cancer-testis antigen NY-ESO-1 and poly-ICLC in Montanide-ISA-51 (Montanide) was found to consistently induce integrated immune responses (antibody, CD4(+), and CD8(+) T cells). Using detailed methods, we investigated the respective effects of poly-ICLC and Montanide adjuvant on pre- and postvaccine NY-ESO-1-specific CD4(+) T cells, because of their central function for induction and maintenance of both antibody and CD8(+) T cells. Polyclonal NY-ESO-1-specific CD4(+) T-cell lines were generated from 12 patients using CD154-based selection of precursors before and after vaccination with (i) OLP alone, (ii) OLP in Montanide, or (iii) OLP and poly-ICLC in Montanide. Kinetics, quantification, fine specificity, avidity, and cytokine-producing pattern were analyzed in depth and compared between vaccine cohorts. Vaccination with OLP alone did not elicit CD4(+) T-cell responses; it suppressed high-avidity CD4(+) T-cell precursors that recognized naturally processed NY-ESO-1 protein before vaccination. Emulsification of OLP in Montanide was required for the expansion of high-avidity NY-ESO-1-specific CD4(+) T-cell precursors. Poly-ICLC significantly enhanced CD4(+) Th1 responses while suppressing the induction of interleukin (IL)-4-producing Th2 and IL-9-producing Th9 cells. In summary, Montanide and poly-ICLC had distinct and cooperative effects for the induction of NY-ESO-1-specific Th1 cells and integrated immune responses by OLP vaccination. These results support the use of admixing poly-ICLC in Montanide adjuvant to rapidly induce antitumor type I immune responses by OLP from self/tumor antigens in human cancer vaccines.

Effects on immunogenicity by formulations of emulsion-based adjuvants for malaria vaccines.

New malaria vaccines are urgently needed to improve vaccine protective efficacy. PfCelTOS is a recombinant malaria vaccine antigen that has shown protective efficacy in a small-animal challenge model when combined with a water-in-oil emulsion adjuvant (Montanide ISA 720). In this report, we show that PfCelTOS vaccines containing GLA-SE (a stable oil-in-water emulsion combined with a Toll-like receptor 4 [TLR4] agonist) elicit strong Th1-type immune responses in BALB/c mice. These responses include higher antigen-specific IgG2a antibody titers and more gamma interferon (IFN-γ) production than those seen with a PfCelTOS vaccine containing Montanide ISA 720. Furthermore, reducing the emulsion dose from 2% to 1% or 0.5% (vol/vol) squalene in GLA-SE did not compromise immunogenicity. Emulsion dose titration in the absence of formulated GLA caused some reduction in humoral and cellular immune responses compared to those with the 2% squalene emulsion dose.

The effect of stimuli on basophil-mediated atopic responses during asthmatic lying-in women and in newborns.

Morbidity from allergic diseases is increasing. Basophils play a critical role in systemic anaphylaxis and chronic allergic inflammation. The prenatal environment must be regarded as a possible early risk factor for allergic diseases in children. Our objective was to determine if basophils harvested from neonates genetically predisposed to atopic disease had different levels of CD63 expression and IL-4 release properties in response to various stimuli (peptidoglycan, Dermatophagoides farinae, hyperosmotic mannitol). Blood samples were collected from 16 asthmatic and 18 healthy women and their newborns. Peripheral blood basophil histamine was measured using the human basophil degranulation test (HBDT), whereas activation was assessed by flow cytometric measurement of CD63 expression on the cord blood basophil surface. IL-4 levels were quantified by ELISA following allergen stimulation. The basophil degranulation index (DI) in granulocytes harvested from the peripheral blood of asthmatic women was assessed following stimulation with peptidoglycan (PGN), Dermatophagoides farinae (Df ) extract, or hyperosmotic mannitol. The DI was significantly higher in atopic women than in healthy controls. Upregulation of CD63 on the cord blood basophil surface was also detected in response to these stimuli. Basophils purified from the cord blood of neonates born to atopic mothers produced more IL-4 compared to basophils purified from the controls. These data suggested that various stimuli play a role in augmenting allergic reactions via modulation of activated basophil cytokine secretion. It may require new methods to stabilize the basophils in allergic diseases.

Phase I trial of Wilms' Tumor 1 (WT1) peptide vaccine with GM-CSF or CpG in patients with solid malignancy.

BACKGROUND: The aim of this study was to investigate the safety and efficacy of combinatorial use of granulocyte-macrophage colony-stimulating factor (GM-CSF) and CpG oligodeoxynucleotides (CpG-ODN) as immunoenhancement adjuvants in Wilms' Tumor 1 (WT1) vaccine therapy for patients with solid malignancy. PATIENTS AND METHODS: The patients were placed into treatment groups as follows: WT1 peptide alone, WT1 peptide with GM-CSF (100 µg) and WT1 peptide with CpG-ODN (100 µg). HLA-A *2402 or *0201/*0206-restricted, WT1 peptide emulsified with Montanide ISA51 was injected intradermally every week for eight weeks. Toxicities were evaluated according to the National Cancer Institute Common Terminology Criteria for Adverse Events ver. 3.0. Tumor size, which was measured by computed tomography, was determined every four weeks. The responses were analyzed according to Response Evaluation Criteria in Solid Tumors. RESULTS: The protocol was well tolerated; only local erythema occurred at the WT1 vaccine injection site. The disease control rate of the groups treated with WT1 peptide alone (n=10), with combinatorial use of GM-CSF (n=8) and with combinatorial use of CpG-ODN (n=10), in the initial two months was 20%, 25% and 60%, respectively. CONCLUSION: Addition of GM-CSF or CpG-ODN to the WT1 peptide vaccine for patients with solid malignancy was safe and improved the effectiveness of clinical response.

A single injection of 19 kda carboxy-terminal fragment of Plasmodium yoelii merozoite surface protein 1 (PyMSP1(19)) formulated with Montanide ISA and CpG ODN induces protective immune response in mice.

OBJECTIVE: To investigate the efficacy of a vaccine formulation of the 19 kDa conserved carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (PyMSP1(19)) formulated with CpG ODN 1826 and Montanide ISA51 or ISA720 when used to immunize mice by a single injection. METHODS: Groups of BALB/c mice were immunized parenterally with one, two or four injections with PBS or PyMSP1(19) formulated with CpG ODN in ISA51 or ISA720. Sera were collected weekly and assessed for total IgG and IgG subclass titers. Protection was tested by challenge infection with P. yoelii YM. RESULTS: Interestingly, single injection immunization showed the same kinetics of antibody responses as two- or four-injection immunization. However, the peak antibody response induced by PyMSP1(19) in CpG ODN and ISA51 appeared earlier than that induced by PyMSP1(19) in CpG ODN and ISA720 (28 days vs 41 days). At day 63 after the first injection, the PyMSP1(19)-specific IgG antibody levels by single injection and four-injection immunizations were not different. However, the levels of the IgG2a antibody subclass were significantly lower by single injection immunization with PyMSP1(19) in CpG ODN and ISA720. The antibodies were sustained at high levels for at least 20 weeks. After challenge infection, all mice immunized by a single injection of PyMSP1(19) in CpG ODN and ISA51 survived with low-grade parasitemia, while 50% of mice immunized with PyMSP1(19) in CpG ODN and ISA720 died with high levels of parasitemia. CONCLUSION: These findings suggest that MSP1(19) immunization by a single injection can induce protective immunity, particularly when formulated with an appropriate strong adjuvant.

Long-term humoral and cellular immune responses elicited by a heterologous Plasmodium vivax apical membrane antigen 1 protein prime/adenovirus boost immunization protocol.

Apical membrane antigen 1 (AMA-1) is an invasion-related Plasmodium antigen that is expressed during both intracellular and extracellular asexual stages of the parasite's life cycle, making it an ideal target for induction of humoral and cellular immune responses that can protect against malaria. We show here that when it is administered as a recombinant protein (P) in Montanide ISA720 adjuvant, followed by a recombinant human type 5 adenovirus (Ad), intense and long-lasting Plasmodium vivax AMA-1-specific antibody responses (including both IgG1 and IgG2a), as well as proliferative memory T cell responses, can be detected in immunized mice. Memory T cells displayed both central (CD44(hi) CD62L(hi)) and effector (CD44(hi) CD62L(lo)) phenotypes, with the central memory phenotype prevailing (56% of AMA-1-specific proliferating cells). Considering the main traits of the memory immune responses induced against AMA-1, this particular sequence of immunogens (P followed by Ad), but no others (Ad/Ad, Ad/P, or P/P), displayed an optimal synergistic effect. These results give further support to the need for preclinical studies of P. vivax vaccine candidate AMA-1 administered in prime/boost protocols that include recombinant proteins and adenoviral vectors.

Mucosal immunity against Eimeria acervulina infection in broiler chickens following oral immunization with profilin in Montanide™ adjuvants.

The present study was conducted to compare aqueous nanoparticle-based Montanide™ IMS 1313 N VG PR (IMS 1313) and oil-based ISA 71 VG (ISA71) adjuvants in combination with an Eimeria subunit protein vaccine on protection against avian coccidiosis. Male broiler chicks were vaccinated twice with an Eimeria recombinant profilin protein alone or in conjunction with IMS 1313 or ISA 71 prior to infection with live, sporulated Eimeria acervulina oocysts. For comparison, chickens were immunized with a commercial live coccidiosis vaccine (Coccivac-B). The following parameters were assessed as measures of protective immunity: body weight gain, fecal oocyst output, profilin-specific intestinal secretary IgA (sIgA) or IgY antibody levels, and percentages of CD4(+), CD8(+), TCR1(+), or TCR2(+) intestinal intraepithelial lymphocytes (IELs). Birds vaccinated with profilin plus ISA 71 had increased body weight gains, equivalent to Coccivac-B vaccination, compared with the profilin-only group. Immunization with profilin plus IMS 1313, or with profilin plus ISA 71, reduced fecal oocysts shedding compared with the profilin-only group. Intestinal sIgA levels were greater in the profilin plus IMS 1313 or ISA 71 groups, and IgY levels were greater in the profilin plus ISA 71 group, compared with profilin alone. Birds vaccinated with profilin plus IMS 1313 or ISA 71 had higher percentages of CD4(+), CD8(+), and TCR1(+), but not TCR2(+), intestinal IELs compared with the profilin-only vaccinated group. Taken together, these results indicate that immunization of chickens with the recombinant profilin subunit vaccine in conjunction with IMS 1313 or ISA 71 adjuvants increases protective immunity against experimental E. acervulina infection.

Adjuvants for enhancing the immunogenicity of whole tumor cell vaccines.

Whole tumor cell lysates can serve as excellent multivalent vaccines for priming tumor-specific CD8(+) and CD4(+) T cells. Whole cell vaccines can be prepared with hypochlorous acid oxidation, UVB-irradiation and repeat cycles of freeze and thaw. One major obstacle to successful immunotherapy is breaking self-tolerance to tumor antigens. Clinically approved adjuvants, including Montanide™ ISA-51 and 720, and keyhole-limpet proteins can be used to enhance tumor cell immunogenicity by stimulating both humoral and cellular anti-tumor responses. Other potential adjuvants, such as Toll-like receptor agonists (e.g., CpG, MPLA and PolyI:C), and cytokines (e.g., granulocyte-macrophage colony stimulating factor), have also been investigated.